Membrane-enclosed transport carriers, such as vesicles, sort biological molecules between stations in the cell in a dynamic process that is fundamental to the physiology of organisms. While much is known about the protein coats that sculpt membranes into vesicles, what remains to be resolved is the mechanism for vesicle release, a membrane fission reaction. In this proposal, we show how a membrane fission mechanism can be dissected by combining powerful biochemical and genetic tools with a versatile and highly sensitive new tool that we have developed: Burst Analysis Spectroscopy (BAS). BAS is a single-particle fluorescence technique that measures changes in particle size and concentration in free solution, allowing detection of intermediates and products during a membrane fission reaction. In the worm model system, C. elegans, release of transport carriers from the signaling organelle known as the recycling endosome, requires a dynamin-like, Eps15-homology domain (EHD) protein, RME-1, functioning with AMPH-1, the worm Bin/ Amphiphysin/Rvs (N-BAR) domain protein. While it is known that cytoskeletal elements, in particular microtubules and actin, participate with vesicle fission machinery in the cell, they are not necessary for membrane fission, in cell-free systems. In vitro, liposome membranes are deformed into rigid, tubular structures, wrapped by the RME-1/AMPH-1 polymer, when locked in an ATP-bound configuration. Although the role of these tubules remains to be discovered, our preliminary experiments with liposomes support a role for RME-1 and AMPH-1 in membrane fission. Here, we seek to reconstitute the minimal fission-active protein machinery in a cell-free assay, to understand how ATP hydrolysis is coupled to membrane binding, rearrangement, and fission. We will further inform our hypotheses for the protein and membrane dynamics required for fission using three-dimensional information provided by additional structural studies performed in collaboration with electron cryo-microscopy and crystallography collaborators. Our hypotheses will be tested in live cells, using genetic mutations in a well-established recycling assay, developed in the C. elegans model system.